Field of the Invention
The present invention relates to a method of manufacturing a patterned substrate for culturing cells, a patterned substrate for culturing cells, a patterning method for culturing cells, and a cell chip; and more particularly, a method of manufacturing a patterned substrate for culturing cells, a patterned substrate for culturing cells, a patterning method for culturing cells, and a cell chip, in which a substrate capable of inhibiting cell adsorption is manufactured using a plasma and cells are selectively cultured by patterning and integrating many numbers of functional groups on the substrate.
Background Information
Researches relating to human life including a human genome project are rapidly increasing. Information analysis and operation of living organisms are gathering strength with ongoing researches on living organisms. Accordingly, an interest in biochips for rapidly analyzing information of organisms has been dramatically increased more than ever before.
Biochips can be divided by a DNA chip, a protein chip and a cell chip according to a biomaterial which is fixed or cultured on a substrate. In the beginning of the biochip era, a DNA chip appeared as a great issue with understanding of human genetic information but protein chips and cell chips have been recently become new attractions of the biochip with the interest in proteins and cells in which a protein binds. Even though non-specificity binding has been an important problem associated with the protein chip, a variety of notable methods are now being introduced.
Among them, the cell chip which is capable of culturing a large amount of cells without affecting their properties is appeared as the most effective tool to access into various fields including novel drug developments, genomics, and proteomics, etc. The cell chip is different from the protein chip in that the rate of cell growth on a substrate in the cell chip is one of indications representing the performance of the cell chip. Meanwhile, when the growth and division of cells cultured on a substrate is observed, the behavior of the cells will be easily analyzed. For example, an effect of cells to a new drug or response of cells to materials in vivo such as hormones can be easily examined.
Various methods for culturing cells on a substrate have been developed, and can be broadly divided by a method using a biomaterial and a method using physical and chemical characteristics of the substrate itself. In the method using a biomaterial, peptides or proteins are first fixed on a substrate, and cells are cultured using cell receptors contained in their biomaterials [Mann B K, Tsai A T, Scott-Burden T, West J L. Modification of surfaces with cell adhesion peptides alters extracellular matrix deposition. Biomaterials 1999; 20(23-24):2281-6].
Examples of the method using physical and chemical characteristics of a substrate include a method using hydrophobic characteristics, a method using electrical characteristics, a method using surface characteristics [Curtis A S, Wilkinson C D. Reactions of cells to topography. J Biomater SciPolym Ed 1998; 9(12):1313-0.29], a method using collagen [On-chip transfection of PC12 cells based on the rational understanding of the role of ECM molecules: efficient, non-viral transfection of PC12 cells using collagen IV, Neuroscience Letters 378 (2005) 40-43], etc.
There are several drawbacks associated with cell chips of which the most general one is that cells are not cultured well on a substrate. When cells are cultured evenly without conglomeration on a substrate, the cells can grow or divide on the substrate. On the other hand, when cells are not cultured properly on the substrate, the cells fail to grow and divide. Further, successful cell culture means that a small amount of cells are exactly cultured. A small amount of cells has to be exactly cultured on a substrate, which can eventually increase the sensitivity of cell chip.
Second, when cells are cultured on the substrate, their intrinsic properties or organization have to be well maintained. If the cells fail to grow or the cells' characteristics are lost due to the substrate even though the cells are well cultured on the substrate, the cell chip cannot perform its function fully. Therefore, it is required to consider the above factors in the development of cell chips.
To solve the above problems, there is a method of efficiently and effectively performing cell culture using a plasma, which is disclosed in PCT/KR2008/001117. This application discloses a method of manufacturing a substrate for fixing cells, a substrate for fixing cells, a method of fixing cells and a cell chip. This application mentions a method of a substrate for fixing cells, a substrate for fixing cells, a method of fixing cells and a cell chip, which is able to fix cells efficiently by integrating many numbers of functional groups on a substrate using a plasma. However, this application mentions only the method of fixing cells on a substrate using a plasma or the like, but does not mention a method of selectively culturing cells by cell adsorption and inhibition of cell adsorption.
When the substrate is patterned to have a surface for inhibition of cell adsorption and a surface for effective cell culture, it can be applicable in the development of implantable chips and artificial organs, genetic experiment, and drug test. However, the application discloses only the method of uniformly culturing cells, and thus it is impossible to selectively culture a small amount of cells in a desired position.
Therefore, the present inventors have studied and invented a method of manufacturing a patterned substrate for culturing cells, which is capable of selectively culturing cells in the desired position of the substrate using a plasma.